In previous work we discovered three classes of spontaneous transient elevations of intracellular calcium (Ca) in embryonic Xenopus spinal neurons and showed that they have distinct functions, regulating aspects of differentiation in a frequency-dependent manner prior to synapse formation: 1) Ca spikes are generated by developmental transient Ca-dependent action potentials and regulate expression of neurotransmitters. 2) Growth cone Ca transients are generated locally in the growth cone and regulate the rate of axon extension. 3) Filopodial Ca transients are produced at the tips of filopodia and regulate growth cone turning. The proposed research has three specific aims that address the mechanisms of action of Ca transients in differentiation of these embryonic spinal neurons. The first aim investigates the mechanisms by which Ca spikes regulate neurotransmitter expression. The second aim analyzes the mechanism generating changes in membrane potential that lead to onset and termination of Ca spiking. The third aim investigates the mechanism by which voltage-gated Ca channels regulate growth cone function. We describe experiments to address the following questions: What molecules are involved in the action of Ca spikes? What mechanisms generate the changes in membrane potential that lead to onset and termination of the period of Ca spiking? Does the mechanism of action of growth cone voltage-gated Ca channels involve interaction with extracellular matrix molecules or mechanically enhanced transmitter release? The immediate goal of this research is to test hypotheses about the mechanisms of Ca transients in the early stages of differentiation of vertebrate spinal neurons. The long term goal is to provide information about the cellular and molecular machinery that governs processes of development, which will contribute to understanding developmental disorders of the nervous system.